1. Field of the Invention
The present invention relates to nucleic acid fragments derived from the genome of an appropriate mycobacterium, in particular Mycobacterium tuberculosis, to their applications in the diagnosis of mycobacterial infections, and to plasmids containing the said fragments.
2. Description of the Background
Mycobacteria correspond to the Mycobacterium genus which comprises at least 54 different species.
Among them, about 10 are pathogenic or opportunistic for man or animals. Two of them, M. tuberculosis and M. Leprae are the agents for tuberculosis and leprosy respectively.
It is known that these two diseases represent a major public health problem; indeed, there are currently between 15 and 60 million people afflicted by tuberculosis worldwide and 2 to 3 million people die each year as a result of this infection. M. tuberculosis is the most common cause of mycobacterial infections in developed countries. In France, 10.sup.4 new cases of tuberculosis appear each year. Vaccination using BCG (Bacille Calmette-Gu erin, an attenuated strain of M. bovis) is far from being effective in the entire population. This efficacy ranges from about 80% in western countries such as England, to 0% in India (results of the last vaccination trial in Chingleput). Furthermore, the appearance of M. tuberculosis strains resistant to the usual anti-tuberculotics and the existence of a correlation between tuberculosis and AIDS adds to the urgency of developing a rapid method of detection and identification of mycobacteria.
For example, an epidemiological study carried out in Florida has shown that 10% of AIDS patients suffer from tuberculosis at the time of the AIDS diagnosis or 18 months before it. In these patients, tuberculosis appears in 60% of cases in a disseminated form which is therefore undetectable by conventional diagnostic tests such as pulmonary radiography or sputum analysis.
Finally, the diagnosis of tuberculosis and other related mycobacterioses is difficult to carry out for various reasons: the pulmonary diseases caused by various mycobacteria cannot be clinically, radiologically or histologically differentiated; mycobacteria are often present at low levels and when they are at levels detectable by the conventional methods used, the disease has already developed and the patients are contagious to others; furthermore, because of the very long generation time in these bacteria (24 h for M. tuberculosis compared with 20 rain for E. coli), the culture of these organisms is difficult. Thus, 6 to 8 weeks are required in order to identify the microbes and more time is required in order to obtain an antibiogram which can be used for the appropriate treatment of the patients. It is therefore essential to be able to have available a detection test which does not require the culture of microbes and which may be used directly with the pathological samples even when the microbes are present therein in low concentrations.
Several techniques are currently used clinically to identify a mycobacterial infection.
First, the direct microscopic detection of the microorganisms should be mentioned; this technique is rapid, but it does not permit identification of the mycobacterial species observed and it lacks sensitivity insofar as a large number of microorganisms must be present in the sample (&gt;10.sup.4 /ml) in order to permit reliable detection (BATES J., CHEST, 1979, 76, (suppl.), 757-763).
The cultures, when they are positive, have a specificity close to 100% and permit the identification of the mycobacterial species isolated; however, as specified above, the growth of the mycobacteria in vitro can only be achieved in 3 to 6 weeks and when few mycobacteria are present at the infection site, repeated cultures are required in order to ensure a correct result (BATES J., 1979 and BATES J. et al., Am. Rev. Respir. Dis., 1986, 134, 415-417).
Serological techniques may prove to be advantageous under certain conditions but their use is limited by their low sensitivity and/or specificity (DANIEL, T. M. et al., Am. Rev. Respir. Dis., 1987, 135, 1137-1151).
The presence or absence of mycobacteria may also be determined by hybridisation to DNA or RNA using probes which are specific for the DNA sequences (KIEHN, T. E. et al., J. Clin. Microbiol., 1987, 25, 1551-1552; ROBERTS, M. C. et al., J. Clin. Microbiol., 1987, 25, 1239-1243; DRAKE, T. A. et al., J. Clin. Microbiol., 1987, 25, 1442-1445). However, these methods are based on the nucleotide sequence polymorphism of the fragments used or on the polymorphism of the adjacent regions and also require the culture of microorganisms.
Some DNA sequences of various mycobacteria and, in particular, some mycobacterial antigen-encoding genes have been described. There may be mentioned in particular International Application PCT WO 88/00974 whose inventor is YOUNG, R. and whose content is taken up in an article which appeared in Nature, 1985, 316, 450; these publications describe the genes encoding five immunodominant M. leprae antigens, and in particular the gene encoding the 65-kDA antigen has been sequenced. International Application PCT WO 88/05823 may also be mentioned, whose co-inventors are HUSSON, R., YOUNG, R. and SHINNICK, T. and whose content is taken up in the article which appeared in J. Bact., 1987, 169, 1080-1088 and which describes the M. tuberculosis genes encoding protein antigens and, in particular, the 65-kDa antigen. This International Application specifies, in particular, that the M. tuberculosis genes encoding five immunologically active proteins were isolated by systematic screening of a recombinant DNA library which is expressed in a bacteriophage lambda gt11, with a collection of monoclonal antibodies directed against the protein antigens of this bacteria. One of the M. tuberculosis antigens, a 65-kDa protein, possesses determinants common to M. tuberculosis and M. leprae.
International Application PCT WO 88/06591, whose co-inventor is in particular T. SHINNICK, describes a 540-amino acid recombinant protein (65-kDa protein) as well as the DNA sequence and the expression vectors of the said protein, and the applications of the said recombinant protein. This application also describes peptides corresponding to sequences of this protein, and their applications.
The genes encoding proteins of other mycobacteria (M. africanum, M. smegmatis, M. bovis BCG and M. avium) have also been isolated. THOLE et al. (Infect. Immunol., 1987, 55, 1466-1475), who have described a 64-kDa H. bovis BCG protein expressed in E. coli, may be mentioned in particular.
However, the amount of mycobacterial DNA present in most biological samples is not sufficient to give a positive signal; the technique of hybridisation therefore proved inappropriate for the detection of mycobacterial DNA extracted directly from biological samples.
A certain number of studies have also shown some structural homology between the various mycobacteria. However, differences in the DNA sequence of M. tuberculosis and M. bovis have been described in the 3' region of the open reading frame of the 65-kDa antigen (SHINNICK et al., 1987, THOLE et al., 1987), but a homologous region has not been observed in M. leprae DNA (MEHRA et al., Proc. Nat. Acad. Sci. USA, 1986, 83, 7013-7017, also PCT 88/000974).
Publications also exist which have demonstrated the existence of repetitive sequences in mycobacteria; in particular, the article by K. D. EISENACH et al. (J. Clin. Microbiol., 1988, 26, 11, 2240-2245) may be mentioned, which describes three cloned segments of M. tuberculosis DNA, which have been identified by selective hybridisation to M. bovis DNA. These three recombinant segments, termed M13KE37 (790 base pairs), M13KE49 (570 base pairs) and M13KE115 (about 1600 base pairs) respectively are obtained by cloning DNA fragments of M. tuberculosis inside the bacteriophage M13. However, this article reveals that the nucleotide sequence of the corresponding segments is not known to its authors.
The article by D. M. COLLINS et al. (FEMS Microbiol. Letters, 1989, 60, 175-178), which describes the identification of a repetitive DNA sequence specific to M. paratuberculosis, may also be mentioned.
Moreover, it is appropriate to mention the article by REDDI et al. (INTERNATIONAL JOURNAL OF LEPROSY, 1988, 56, No. 4, p. 592-598 ) which describes the existence inside the DNA of Mycobacterium tuberculosis H37Rv and H37Ra, of repetitive fragments whose sequence has not been determined. The repetitive fragments described by REDDI et al., which are 5.6 and 4.8 kb fragments respectively, are not specific to the tuberculosis bacillus group since they are also present in M. kansasii which does not belong to the tuberculosis complex.
The following additional references also constitute the state of the art prior to the present invention.
BAESS, I., Acta Path. Microbiol. Scand., 1979, 87, 221-226; BEAUCAGE, S. L. et al., Tetrahedron Lett., 1981, 22, 1859-1862; EISENACH, K. D. et al., Am. Rev. Respir. Dis., 1986, 133, 1065-1068; GHEORGHIU, M. et al., J. Biol. Standardization, 1988, 16, 15-26; GLASSROTH, J. et al., N. Engl. J. Med., 1980, 302, 1441-1450; HAWKINS, C. C. et al., Ann. Intern. Med., 1985, 105, 184-188; IMAEDA, T., Int. J. Systematic Bacteriol., 1985, 35, 147-150; IMAEDA, T. et al., Int. J. Systematic Bacteriol., 1988, 38, 151-156; KOGAN, S. C. et al., N. Engl. J. Med., 1987, 317, 985-990; LI, H. et al., Nature (Lond.), 1988, 335, 414-417; LU, M. C. et al., Infect. Immun., 1987, 55, 2378-2382; MANIATIS, T. et al., 1982, Cold Spring Harbor, N. Y.; MCFADDEN, J. J. et al., Mol. Microbiol., 1987, 1, 283-291; PAO, C. C. et al., Tubercle, 1988, 69, 27-36; PATEL, R., J. Gen. Microbiol., 1986, 132, 541-551; SAIKI, R. K. et al., Science, 1988, 239, 487-491; SANGER, F. et al., Proc. Natl. Acad. Sci. USA, 1977, 74, 5463-5467; SMIDA, J. et al., Int. J. Leprosy, 1988, 56, 449-454; THEIN, S. L. et al., in Human Genetic Diseases, 1986, IRL Press, 33-50; THOLE, J. E. R. et al., Infect. Immun., 1985, 50, 800-806; WATSON, E. A., Canad. J. Pub. Health, 1935, 26, 268-275; WOLINSKY, E., Am. Rev. Respir. Dis. 1979, 119, 107-159.
Recently, a method of detection of low amounts of mycobacteria by amplification and hybridisation directly on biological samples has been developed; the said method uses the nucleotide sequence polymorphism of a gene fragment common to all mycobacteria, and in particular a fragment of the gene encoding the 65-kD protein (French Patent Application No. 89 05057).